Bioelectricity production using double chambered Microbial Fuel Cell (MFC)
Abstract: Finding an alternative renewable resource of energy is the need of the hour. The Microbial Fuel Cell technology is a new avenue acting as an alternate source of energy which uses various forms of waste (industrial, kitchen or domestic waste etc.) for the generation of electricity, converting chemical energy into electrical energy using a bio-electrical system. A total of six potential isolates were obtained for bioelectricity production. A pilot scale bioreactor study revealed the potential of isolate VITSDPJ4 for electricity production. A minimal media supplemented with powdered peels of orange was used as a substrate for the production of bioelectricity and current produced was found to be 290µA by VITSDPJ4. Biochemical and 16S rRNA analysis was carried out to identify the effective isolate VITSDPJ4.
Index terms: Microbial fuel cells, bioelectricity, 16S rRNA, Substrate.
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Increasing problems of recent rise in energy costs, supplies of energy sources and parallel complications like pollution and global warming have reignited the interest in finding a cheap alternative renewable energy source.
In order to mitigate this crisis, a cheap alternative source is required to balance the depleting fossil fuel reserves [1, 3]. Treatment and recycling of wastes is crucial issue due to the limitation of disposal sites . Large volumes of organic wastes and domestic waste are being generated every day from breweries, dairy, food processing industries, livestock waste. Microbial production of electricity is an emerging form of bioenergy since it offers the possibility of generating current from a wide range of complex organic wastes [3, 4].
MFC (Microbial Fuel Cell) is a bio-electrochemical transducer for converting the chemical energy stored in organic waste to electrical energy by the help of potential microorganisms. Based on the mechanism of transfer of electrons from bacteria to electrode, MFCs are basically classified into two types, mediator (methyl blue, methyl viologen) and mediatorless MFCs . In the anode chamber of MFC bacteria oxidizes the substrate anaerobically and generates electrons and protons. The electrons generated are transferred from the anode to cathode through an external circuit and protons diffuse through the agar salt bridge [6, 7, 9 and 11]. The power generation capacity of MFC depends on factors such as electrodes used in the anode and cathode chamber, substrate or carbon source used, presence or absence of mediators, nature of inoculum and electrolyte used in the cathode chamber.
Geobacter sulfurreducens has previously been reported to have the potential of producing high densities of electricity in MFC’s [4, 8]. In addition to Geobacter, other microbes predominantly capable of bioelectricity generation includes Clostridium acetobutylicum, Clostridium thermohydrosulfuricum, Saccharomyces cereviseae etc. and a few marine microalgae . Citrus juice industries produces enormous quantities of processing residues and the disposal by physical methods such as incineration is energy consuming and also contributes to environmental pollutions. Hence using these wastes as substrate in bioelectricity production could be an effective alternate .
The current study focuses on the isolation of bioelectricity producing bacteria from soil and cheap industrial waste such as bagasse, molasses .Orange peels was used as a substrate at varied concentrations for the scale up bioreactor assay.
II. MATERIALS AND METHODS
- Sample collection
Samples like soil, bagasse and molasses were procured from a sugar mill situated in Vellore district. The substrate orange peel was obtained from a local market in Vellore, Tamil Nadu.
- Isolation of bacteria
For the isolation of bioelectricity producing organism the samples were serially diluted and 10-3 was plated onto Luria Bertani Agar. The plates were incubated at 30° C for 24h. The visible colonies obtained were purified and was preserved in glycerol stock.
- Processing of substrate
The orange peel was washed with tap water and dried in sunlight. The dried substrate was ground into fine powder using mortar and pastel and was stored at 25°C. 20g of powdered orange peels used as substrate was added to the anodic chamber containing 300 ml MSM.
- Media preparation and optimization
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Minimal Salt Medium having the composition Na2HPO4.12H2O (11.2 g/l), KH2PO4 (2.4g/l), (NH4)2SO4 (2g/l), MgSO4.7H2O (200mg/l), MnSO4.4H2O (10mg/l), Nacl (10mg/l), CaCl2 (10mg/l), ZnCl2 (5mg/l) and CuSO4 (0.5mg/l)  was prepared and 20g of orange peel was added as a carbon source, pH was adjusted to 7.
- Construction of MFC
Copper electrodes were used at both ends of cathode and anode chambers containing KMnO4 and the culture broth respectively. A copper wire was used to connect the electrodes to the multimetre [6, 7 and 12].
- Anode chamber
One litre plastic bottle was used which was surface sterilized using 70% ethanol and 3% sodium hypochloride solution, rinsed with sterile distilled water followed by UV exposure for 20 min. The medium containing substrate and inoculum was filled in it. This chamber was maintained in anaerobic condition .
- Cathode chamber
An aerated 1L surface sterilized plastic bottle containing 0.5mM KMnO4 was used as cathode.
- Salt Bridge
A salt bridge was prepared, dissolving 6g Nacl and 5g of Agar Agar in 150ml of distilled water. This was transferred into surface sterilized PVC pipe, and kept overnight in the refrigerator. This system was used to complete the circuit by connecting the two half cells.
- Circuit assembly
The two chambers were connected externally by copper wires (Fig.1).The MSM media containing the effective isolate VITSDPJ4 was inoculated in the bioreactor and the amount of electricity generated was assessed at an interval of one hour for 24h.
Fig. 1. Diagrammatic representation of MFC
- Identification of effective isolate
- Morphological and Biochemical characterization
Various biochemical characterization was performed according to Bergey’s Manual like Gram’s staining, IMViC, motility and catalase for the identification of VITSDPJ4.
- Molecular characterization
The characterization of the effective strain was performed by amplification of 16S rRNA sequence using universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). The DNA was extracted from cells and the 16S rRNA was sequenced by the fluorescent dye terminator method using the sequencing kit (ABI Prism Big dye terminator cycle sequencing ready reaction kit v.3.1). The product being run on a ABI13730XL capillary DNA sequencer (ABI Prism 310 genetic analyser, Tokyo, Japan). The sequence there by obtained were aligned using ClustalW software and phylogenetic tree was constructed by neighbour join method employing mega4 software.
- Isolation of bioelectricity producing bacteria
A total of six morphologically distinct isolates were obtained and purified on Luria Bertani agar plates. The soil isolates especially VITSDPJ4 was found to be fast growing compared to others, so VITSDPJ4 was selected as an effective isolate.
- Medium preparation and optimization
For the optimization, 100 ml of minimal salt medium was prepared and 0.6gm of the powdered orange peel was supplemented as substrate, further with 2% of 48h seed culture of VITSDPJ4 was inoculated. The electricity produced was recorded using a multimetre every hour for minimum 12h. (Fig.2 and Fig.3)
Fig.2. Construction of MFC
The amount of current generated was meagre so to increase the efficiency of the bioreactor optimization of the media and substrate was performed by increasing the quantity. 300 ml of MSM was prepared and 10g and 20g of orange peel powder was added as substrate, 2% of 48h inoculum of VITSDPJ4 isolated from soil was added. The electricity produced was recorded using a multimetre every hour for minimum 12h (Fig.3).
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Fig. 3. Graph representing bioelectricity production by VITSDPJ4
- Identification of effective isolate
- Morphological and Biochemical characterization
Various morphological and biochemical characterization revealed effective isolate VITSDPJ4 to be gram negative bacilli, catalase positive, non-motile and IMViC negative.
- Molecular characterization Results awaited.
In the present study bioelectricity production from double chambered MFC was performed to identify an alternative to the electricity production from fossil fuels. According to Hernandez et al., 2013, it has been demonstrated that the mediator-less MFC could be used to generate an electrical current, and its performance was monitored through long operation using buffered LB medium as the nutrient source and copper electrodes.
The Bioreactor in the present study consisted of an anode and cathode chamber with copper electrodes and was supplemented with the substrate and catholyte respectively. The medium supplemented in the anode chamber consisted of micronutrients and peel powder was supplemented as carbon source. The bacterial isolate VITSDPJ4 showed the faster growth was considered to be the effective isolate. It was then inoculated individually by altering the concentration of the substrate in each batch fermentation which was done in order to evaluate the role of substrate in bioelectricity production. Microorganism obtain energy by oxidizing organic matter and thus convert the chemical energy into electrical energy . The electrons generated during bacterial metabolism at the anode chamber are transferred to the cathode via an external circuit and the protons diffuse through the salt bridge that connects the two halves of the cell [6, 7, 9 and 11]. The electricity production in double chambered MFC can be attributed to the utilization of orange peel powder as substrate by the bacterial isolate and its subsequent decomposition. Maximum and consistent electricity production was observed when substrate concentration was 20g at 11th h after inoculation suggesting effective electron discharge by the organism due to optimal metabolic activity and degradation of the substrate. Various factors attribute to the intensity of electricity generated in MFC. The density of electricity generated may depend upon the extent of colonization of the anode chamber by the bacterial isolate. Pre-treated wastes fastened the release of dissolved organic matter which in turn contributed greater electricity production .During the third run of the bioreactor supplemented with 20g of orange peel powder although maximum power generation of 0.27 mA was obtained the electricity generated declined by the 12th hour which may be due to the release of organic acids as part of the normal metabolic activity of the organisms and by virtue of the acidic content of the substrate peel. The pH of the entering substrate also effects the environment at the anodic chamber for microbial growth which should be between 6.5 and 7, thus maintaining the pH by addition of H2SO4 or NaOH will allow consistent growth of the organism and efficient power generation. The research work by Gil et al., 2003, Zhen He et al., 2008; and Jadhav et al., 2009, has proved that power generation by microorganisms occurs optimally at pH of 7 since microbial activity at a pH lower than the optimal pH is always low [17, 18 and 19]. In this study a batch bioreactor was employed wherein the substrate and the inoculum was fed into the reactor only once which limits us from exploiting the potential of organism which could be the main reason for low electricity generation. Using a consortium that possesses faster generation time will help in generating more power output due to quicker achievement of optimal cell density for steady flow of electrons from anode to cathode . As shown by Qing Wen et al., 2009; and S. Venkata Mohan et al., 2008; use of continuous fed batch system prevents the unavailability of substrate by providing a continuous supply thereby maintaining a suitable density of active cells leading to a consistent output of current [20, 21]. This study demonstrates the feasibility of MFC and opens an avenue for remediation and better use of fruit peel waste. Many such systems can be connected in series with a view to scale up the electricity generated which has a promising future for fruit processing industries.
From the present study it could be concluded that the citrus fruit peels shelled out by fruit processing industries can be used as a substrate for effective electricity production by microorganisms as biocatalyst. Thus, this system acts as an economical renewable alternative source for electricity production.
The authors wish to thank the lab members of VIT for their contribution.